CN101340930A

CN101340930A - Method for inactivating pathogens in donor blood, plasma or erythrocyte concentrates in flexible containers in motion

Info

Publication number: CN101340930A
Application number: CNA2006800479151A
Authority: CN
Inventors: 哈拉尔德·莫尔
Original assignee: Blutspendedienst der Landsverbande des Deutschen Roten Kreuses
Current assignee: Blutspendedienst der Landsverbande des Deutschen Roten Kreuses
Priority date: 2005-12-23
Filing date: 2006-12-21
Publication date: 2009-01-07
Anticipated expiration: 2026-12-21
Also published as: US8164073B2; EP1962905A1; ES2341368T3; CN101340930B; KR20080081038A; EP2198886A1; JP5232008B2; US20070164233A1; ES2369059T3; DE502006006204D1; HK1121406A1; HK1121407A1; WO2007076832A1; DE102005062634A9; AU2006332291B2; PT2198886E; DE102005062634A1; CA2632558A1; DK1962905T3; PL1962905T3

Abstract

The invention relates to a method for inactivating pathogens, such as bacteria and viruses, in donor blood, blood plasma and erythrocyte concentrates by photodynamic treatment and/or irradiation with ultraviolet radiation in flexible exposure bags under intense movement.

Description

The method of pathogen in deactivation donor blood, blood plasma or the erythrocyte concentrate under in flexible container, moving

Technical field

Theme of the present invention is a pathogen in deactivation donor blood (blood), blood plasma (blood plasma) and/or the erythrocyte concentrate (EK), the method for antibacterial and virus for example, and described method is handled by light power and/or is utilized ultraviolet radiation to carry out.

Background technology

Be known that the application in treatment of blood and blood products has risk, i.e. receptor infective virus and antibacterial.For example Type B viral hepatitis (HBV) and hepatitis C (HCV) and acquired immune deficiency syndrome (AIDS) pathogen HIV-1 and HIV-2.If do not carry out the step of deactivation or elimination pathogen when the preparation corresponding articles, this class risk exists always.

In the past and now all do a lot of effort, by the light dynamic method goods that purify the blood.Principle is based on related product is exposed in the presence of photoactive substance (photosensitizer).The light of injecting blood products must comprise such wave-length coverage, and described wave-length coverage can and can activate photosensitizer by sensitiser absorption.Absorbed energy or directly transfer to related objective structure (for example Bing Du nucleic acid or surface protein) destroys object construction thus, perhaps transfers on the dissolved oxygen molecule, thus the excited oxygen molecule.Produce singlet oxygen, it has and kills the virus significantly and antimicrobial activity.

Used photosensitizer has high-affinity to the virus and the basis of other pathogen ideally, for example to their nucleic acid, and the goods composition that should purify is had only very low or does not have affinity at all.The result who handles as photodynamics, inactivating pathogens in this case, and retained product activity.As the suitable photosensitizer of handling blood plasma phenothiazine dyestuff methylene blue has for example been described; Riboflavin (vitamin B2) is used for purifying platelet concentrate, and has tested phthalocyanine dye and purify EK.Yet the method that is used for light power deactivation EK pathogen does not also exceed laboratory scale up to now.

For blood itself even all the more so.A main cause may be, the light of being injected must have certain intensity, thereby can activate used photosensitizer, and blood and EK only have extremely low penetrance to the light of each wavelength.Also there is same problem certainly in blood plasma, although the degree difference.

It is also known that only by the radiation of shortwave ultraviolet (UV) light, promptly in about wave-length coverage of 200 to 320nm, especially 200 to the wave-length coverage less than 300nm (UVB and UVC), equally can inactivating pathogens.It is too low to surpass the then radiating energy of 320nm, can not deactivation microorganism and virus.Opposite with the photodynamics method with pathogen inactivated chemistry, photochemistry, only the radiation with UV light has itself separately effectively basically, and the advantage that does not need to add reactive chemicals or photoactive substance.

It is the most effective being used for direct pathogen inactivated UVC.Yet it also has shortcoming, and promptly it penetrates and contains protein solution for example a blood plasma or cloudy suspensions (for example blood and EK) to the utmost point low penetration depth.UVC is used to carry out the sterilization of blood plasma and albumin solution, especially deactivation hepatitis virus during World War II and thereafter soon.Program at that time is that solution passes through the UVC light source as thin film in flowing through device.Described method is proved safe inadequately and is abandoned (Kallenbach NR, Cornelius PA, Negus D, et al.Inactivation of virusesby ultraviolet light.Curr.Stud.Hematol.Blood Transfus.1989,56,70-82).

Use improved method at present, to treat sterilization with plasma protein products according to identical principle operation.In all cases, all be to handle larger volume with present purpose in the past, it is blood plasma storehouse or up to several hectolitres or more protein solution (Hart H.Reid K, Hart W.Inactivation of viruses during ultraviolet light treatment of humanintravenous immunoglobulin and albumin.Vox Sang 1993; 64 (2): 82-88und Chin S.Williams B, Gottlieb P, et al.Virucidal short wavelengthultraviolet light treatment of plasma and factor VIII concentrate:protection of proteins by antioxidants; Blood 1995; 86 (11): 4331-4336).

Be used to sterilize a plurality of volumes in each case up to single unitary blood supply, blood plasma or the EK of hundreds of milliliter, above-mentionedly flow through device and be not suitable for.Yet this is blood bank actual needs every day just.

UVB be microbicide be again antiviral, although compare on degree different with UVC.UVB penetrates and contains protein solution and muddy suspension is better slightly than UVC, yet, its penetration depth, for example the penetration depth in blood plasma is also only in several millimeters scope.

Blood plasma separates from single blood supply with the EK great majority, perhaps also obtains by machine list blood sampling composition art from single donor.The volume of goods is generally about 200 to 350ml.The volume great majority of blood supply are 450 to 500ml.Described goods freeze (blood plasma) in the flat, plastic bag usually deeply or (blood supply EK) is stored at about 4 ℃.

What expect is in this class bag described goods to be sterilized.Yet, there are the problems referred to above at this, promptly it almost is an impermeability to UV light, in addition, blood and EK are impermeabilities to visible light.

Find that unexpectedly top problem is solved by the method according to claim 1.Embodiment preferred is the theme of dependent claims, perhaps describes hereinafter.

Summary of the invention

According to the present invention, make goods, promptly donor blood (blood), blood plasma (blood plasma) and/or erythrocyte concentrate (EK) move in the mode that is fit in its exposure bag, thereby realize sample circulation continuously in container.Described motion is so strong at this, makes that described layer is so thin, feasible can being penetrated by light that used at liquid or the zonal layer of the inner formation of suspension.Described motion must make simultaneously that solution or the suspension in the bag effectively mixed.If satisfy following prerequisite, then the both is achieved:

1. described exposure bag highly flexible, and between exposure period, do not fix described exposure bag, for example be not clipped between glass plate or the quartz plate.The blood plasma that is produced when therefore, the described exposure bag of its adaptation moves or the change of shape of suspension (blood, EK).

2. described exposure bag is filled to maximum 30%, especially maximum 15% of maximum packing volume.

3. make described bag strong movements, for example level (linear back and forth or circular or oval) or vertical (waving) motion.

Strong movements is interpreted as in this (separately or in combination):

1. it has exceeded and has only caused liquid or the blended motion of suspension.

2. in the liquid or the suspension inside of strong movements, temporary transient at least even form the zone on different positions, described zone is so thin, makes UV light or visible light (latter is applicable to muddy or coloured liquid or suspension, for example EK) can penetrate it.

3. the reverse of the direction of motion is so unexpected during strong movements, makes the major part of goods described in the exposure bag because inertia continues to original direction motion, so remainder can form thin layer, and this thin layer can be penetrated by light that used.

In conjunction with the continuous mixing of carrying out simultaneously, make whole goods (with the pathogen that wherein comprises) exposure at last, thus with its sterilization.

Described exposure bag has only several mm thick under the occupied state of level, and for example less than 10mm, preferably less than 5mm, and for example 200 to 500ml sample volume is held in plan.Yet the heap(ed) capacity (volume) of described exposure bag is at least 3 times of the actual pending sample volume that comprises wherein, at least 6.66 times usually, and preferably at least 10 times or even at least 20 times.

Experimental study

The effectiveness of described description of test method, but be not limited to the deactivation of described virus.For use in the described experiment from the blood plasma of blood supply or EK also without limits, the goods that the method according to this invention can also be used for otherwise preparing.The experiment of all descriptions has all been carried out 3 to 6 times.The result who provides represents the meansigma methods of these experiments respectively.

Blood plasma unit and erythrocyte concentrate

Used blood plasma unit and EK prepare from single blood supply by common method.Its volume is about 250 to 300 or up to 350ml, and is kept at the common plastics bag that is used for blood products.Remaining leukocyte or platelet are by removing by filter.EK is suspended among the stabilizing agent medium SAG-M.Blood plasma is being lower than under-30 ℃ the temperature and is storing, and melts in water-bath when being used for testing.EK is stored in 4 to 8 ℃ the cold room.

Virological investigation

The aliquot of blood plasma or EK is mixed vesicular stomatitis virus (VesicularStomatitisvirus) (VSV respectively, the Indiana strain, ATCC VR 158) or sindbis virus (Sinbis-Virus) (ATCC VR-68) or herpesvirus suis (Suid Herpesvirus) (SHV-1, pseudorabies virus, the Aujeszky strain, ATCC VR-135).Determine virus titer by CPE method of testing (CPE=CPE).With TCID ₅₀(TCID=TCID) provides.African green monkey kidney cell strain system (Vero) cell is as indicator cells.Original virus concentration is about 10 in the experiment of being implemented ⁵To 10 ⁷TCID ₅₀

Exposure device, the exposure bag

Used exposure device is equipped with emission UVC light (wavelength: pipe 254nm).Radiation is carried out from the both sides of the exposure bag placed, promptly from top and below.Second exposure device is equipped with the pipe of emission UVB light (280 to 320nm).Radiation is carried out from both sides equally.The 3rd exposure device is equipped with the LED (light emitting diode) of the strong HONGGUANG of emission wavelength ranges about 635nm.All three kinds of devices are in operation and are placed on per minute up to 100 orbit oscillator (Buehler company of manufacturer, the Tuebingen that change; SM 25 types) on.Used exposure bag is made by thin UV penetrance and high flexibility plastic foil.

EXPERIMENTAL EXAMPLE 1

By the deactivation of UVC: the influence of the free activeness of blood plasma between concussion speed and radiation era to VSV in the blood plasma

In the blood plasma unit in the exposure bag, mix VSV, and with UVC radiation 2 minutes.A sample shakes under 100rpm, and is being clipped in securely between radiation era between two quartz plates.Other sample only is placed on the quartz plate, so that during the earthquake can move in the inside of bag.The rotating speed of oscillator changes between 30 to 100rpm.Summed up the result in the table 1.In the sample of firmly clamping, virus titer has only reduced about 0.3log ₁₀Coefficient.

Table 1

Concussion frequency (rpm)	Virus titer (log ₁₀TCID ₅₀)	Note
Concussion frequency (rpm)	Virus titer (log ₁₀TCID ₅₀)	Note	0	6,21±0,69	Contrast
30	5,78±0,27	Loose placement	0	6,21±0,69	Contrast
30	5,78±0,27	Loose placement	50	4,59±0,04	Loose placement
75	0,92±0,24	Loose placement	50	4,59±0,04	Loose placement
75	0,92±0,24	Loose placement	100	0,35±0,52	Loose placement
100	5,92±0,11	Clamp	100	0,35±0,52	Loose placement

For the sample of loose placement, rotating speed has direct influence to the degree of inactivation of virus: rotating speed be 30 or 50rpm during, it is about 1.1 or 2.4log that the deactivation coefficient is compared with untreated control sample ₁₀, when 75rpm, rise to approximately 5.1, when 100rpm, rise to about 6.6log ₁₀This result of experiment has proved that blood plasma must shake strongly, thereby can make the UV light radiation become efficient during handling.In order to make that shaking effect also works, the necessary loose placement of sample, thereby the thin layer that formation can be penetrated during the earthquake.

EXPERIMENTAL EXAMPLE 2

Radiation by utilizing UVC is to the deactivation of VSV, sindbis virus and SHV-1 in the blood plasma:

Deactivation kinetics

In the blood plasma unit, mix VSV, sindbis virus or SHV-1, and radiation 1 to 5 minute.The loose sample that is placed on the orbit oscillator moves with 100rpm, radiation control sample 5 minutes, but do not shake.Summed up experimental result in the table 2.In the sample through concussion, the titre of VSV reduced greater than 6.5log in 3 minutes ₁₀Coefficient, and not the concussion control sample in, the deactivation coefficient is no more than 1.5log ₁₀It is more stable than VSV that the sindbis virus is proved to be; But, demonstrated big difference once more between the sample through concussion and not concussion: after 5 minutes radiated time, reduced about 5.1log through virus titer in the sample of concussion at this ₁₀, and in the sample of not concussion, only reduced 0.30log ₁₀Also obtained similar result when using SHV-1: in 4 to 5 minutes, reduced 4.3 to 4.5log through virus titer in the sample of concussion ₁₀Coefficient, and radiation has only reduced 0.3log after 5 minutes in the sample of not concussion ₁₀

Table 2

EXPERIMENTAL EXAMPLE 3

Radiation by utilizing UVB is to the deactivation of VSV in the blood plasma: deactivation kinetics

In the blood plasma unit, mix VSV, and radiation 1 to 5 minute.The loose sample that is placed on the orbit oscillator moves with 100rpm.Radiation control sample 5 minutes, but do not shake.As seen from Table 3, in the sample through concussion, virus titer reduced 6.36log in 5 minutes ₁₀Coefficient, and in the control sample of not concussion, only reduce about 1.5log ₁₀The result shows, the phenomenon of being found (making pathogen inactivated rising in the sample of loose placement by strong concussion) is not only limited to UVC.

Table 3

UVB (minute)	Concussion	Virus titer (log ₁₀TCID ₅₀)
UVB (minute)	Concussion	Virus titer (log ₁₀TCID ₅₀)	Contrast	-	7,00±016
2	+	4,70±0,08	Contrast	-	7,00±016
2	+	4,70±0,08	3	+	3,68±0,12
4	+	2,23±0,23	3	+	3,68±0,12
4	+	2,23±0,23	5	+	0,64±0,08
5	-	5,52±0,08	5	+	0,64±0,08

EXPERIMENTAL EXAMPLE 4

Handle deactivation by the light power that utilizes methylene blue and light to VSV in the blood plasma

In the blood plasma unit, mix VSV, sneak into 0.25 μ M/L photosensitizer methylene blue (MB) and be to utilize the red LED light radiation to be thirty minutes long most on the orbit oscillator of 100rpm at revolution.Control sample exposed 20 minutes in the presence of same concentration MB, but did not move during this period.As shown in table 4, the inactivation of virus degree is much larger than not shaking sample in the process concussion sample.Latter's virus titer after 20 minutes reduces about 4.4log ₁₀Coefficient; Reduce about 5.8log after 30 minutes ₁₀In contrast, after 20 minutes, reduce coefficient in the sample that does not move and be not more than about 2.7log ₁₀

Table 4

MB/ light (minute)	Concussion	Virus titer (Log ₁₀TCID ₅₀)
MB/ light (minute)	Concussion	Virus titer (Log ₁₀TCID ₅₀)	Contrast	-	6,72±0,24
10	+	4,95±0,68	Contrast	-	6,72±0,24
10	+	4,95±0,68	20	+	2,30±0,88
30	+	0,94±0,87	20	+	2,30±0,88
30	+	0,94±0,87	20	-	4,04±0,54

EXPERIMENTAL EXAMPLE 5

Handle deactivation by the light power that utilizes methylene blue and light to VSV among the EK

In the EK aliquot, mix VSV, sneak into 5 μ M/L photosensitizer methylene blues (MB) and be to utilize the red LED light radiation to be thirty minutes long most on the orbit oscillator of 100rpm at revolution.In contrast, control sample does not move between exposure period.Table 5 shows tangible experimental result.Clearly, through the concussion the EK sample in inactivation of virus greatly faster than not shaking the EK sample.In the sample that in processing procedure, moves, viral (the deactivation coefficient 6.7log that after 30 minutes, almost completely is inactivated ₁₀).In contrast, after 30 minutes, do not reduce the coefficient 2.7log that only has an appointment in the sample of motion ₁₀

EXPERIMENTAL EXAMPLE 4 and 5 result proved, if sample is shaken between exposure period strongly, then also improves the efficient that the light power of blood plasma or erythrocyte concentrate is handled greatly.

Table 5

MB/ light (minute)	Concussion	Virus titer (Log ₁₀TCID ₅₀)
MB/ light (minute)	Concussion	Virus titer (Log ₁₀TCID ₅₀)	Contrast	-	7,04±0,26
10	+	2,62±0,31	Contrast	-	7,04±0,26
10	+	2,62±0,31	20	+	0,89±0,21
30	+	0,30±0,12	20	+	0,89±0,21
30	+	0,30±0,12	10	-	5,07±0,26
20	-	5,25±0,31	10	-	5,07±0,26
20	-	5,25±0,31	30	-	4,35±0,27

Claims

1. A method for inactivating pathogens in donor blood, blood plasma and/or erythrocyte concentrate, said method comprising the steps of:

- provision of blood supplies or products obtained from donor blood and/or by mechanical apheresis,

or

(b) irradiating said article with ultraviolet (UV) light having a wavelength of 200 to 320 nm,

- wherein the article consists of a number of units which can be handled individually and stored separately, and

- said article is located in a flexible light-transmissive (Variant (a)) or UV-transmissive (Variant (b)) flat exposure bag, respectively,

It is characterized in that,

- said exposure pocket is filled with less than 30% by volume of the maximum fill volume of the exposure pocket, and

- moving the exposure pockets during the photodynamic treatment and/or irradiation with UV light in such a way that the contents of the exposure pockets are circulated and zones of varying layer thickness are formed by the movement.

2. The method according to claim 1, characterized in that the pathogen is a virus and/or a bacterium.

3 . The method as claimed in claim 1 , characterized in that, as a result of the movement, the strip has regions which regularly and temporally acquire a layer thickness of less than 1 mm, preferably less than 0.05 mm. 4 .

4. Method according to at least one of the preceding claims, characterized in that the movement, in particular the magnitude of the movement, is effected such that within the product there are regions with layer thicknesses which are regularly and temporarily smaller than 0.05 mm.

5. The method according to at least one of the preceding claims, characterized in that the sum of the areas of the bottom side and the top side of the exposed bag which are in contact or can come into contact with the bag contents is greater than 90% of the total inner surface area of the bag contents Percentage, preferably greater than 99 area percent.

6. The method according to at least one of the preceding claims, characterized in that, in the case of embodiment (b), the radiation is or comprises UVB less than 320 nm to 280 nm and/or UVC less than 280 nm to 200 nm, in particular less than The UVC from 260 nm to 220 nm preferably consists exclusively of radiation having wavelengths in the above-mentioned range.

7. The method according to at least one of the preceding claims, characterized in that each unit originates from one to six donors, in particular from one donor.

8. The method according to at least one of the preceding claims, characterized in that the irradiation is carried out with UVB having a light energy of 0.3 to 10 J/cm ² , preferably 0.5 to 5 J/cm ² .

9. The method according to at least one of claims 1 to 7, characterized in that the irradiation is carried out with UVC having a light energy of 0.01 to 5 J/cm ² , preferably 0.1 to 2 J/cm ² .

10. Method according to at least one of the preceding claims, characterized in that the exposure bag has a volume of at most 5000 ml.

11. Method according to at least one of the preceding claims, characterized in that the exposure bag is held movably in the device and in particular is not sandwiched between two planes, the exposure bag being moved in the device and is irradiated.

12. Method according to at least one of the preceding claims, characterized in that the exposure bag is in motion for at least three quarters, in particular at least five sixths, of the total exposure duration.

13. Method according to at least one of the preceding claims, characterized in that the exposure bag is moved by oscillating, tilting and/or by rotating.

14. The method according to at least one of the preceding claims, characterized in that the product is plasma and consists of more than 80% by weight of blood plasma.

15. The method according to at least one of claims 1 to 13, wherein the preparation is an EK preparation and has a hematocrit of 10 to 75% by weight, preferably 20 to 60% by weight.

16. The method according to at least one of the preceding claims, characterized in that the photosensitive substance is one or more phenothiazine dyes, especially thionine, methylene blue, toluidine blue and/or azure dyes A, B and C.

17. The method according to at least one of claims 1 to 15, characterized in that the photosensitive substance is one or more phthalocyanine compounds.

18. The method according to at least one of claims 1 to 15, characterized in that the photosensitive substance is one or more porphyrin compounds.

19. Method according to at least one of claims 16 to 18, characterized in that the photodynamic treatment is carried out only with wavelengths in the absorption range (+/- 20 nm) of the photosensitizer or agents used.

20. Method according to at least one of the preceding claims, characterized in that the exposure pocket is filled up to a maximum of 15% of the maximum filling volume.

21. The method according to at least one of the preceding claims, characterized in that the blood plasma is processed according to step (a) in the absence of photosensitizers.

22. The method according to at least one of the preceding claims, characterized in that the shaking is carried out with an orbital shaker, a platform shaker, a rocking shaker or a tumble shaker.

23. Method according to at least one of the preceding claims, characterized in that the exposure bag is placed on one side, so that during or by movement or oscillation, the height of the exposure bag is constantly changed, the height The entire upper surface of the exposure pocket is considered based on the distance (surface normal) between the plane on which the exposure pocket is placed and the point of intersection with the upper surface of the exposure pocket.

24. The method according to at least one of the preceding claims, characterized in that the exposure pockets have an average fill height of less than 5 mm and that troughs are continuously produced by movement, resulting in a layer thickness of less than half the average fill height, preferably less than 1 mm Or even layer thicknesses of less than 0.05 mm.

25. The method according to at least one of the preceding claims, characterized in that the exposure bag is made to be continuously larger than An amplitude movement of 0.2 mm, preferably greater than 1 cm, in particular 1 to 15 cm or 2 to 8 cm, and independently of this, a frequency of direction change of said movement greater than 0.5 Hz, preferably 1 to 10 Hz.